During the project, wood samples for metagenomic analysis of dead wood from eight tree species were collected from a mass windfall site in a mixed broadleaved forest: Quercus robur, Fraxinus excelsior, Tilia cordata, Ulmus glabra, Acer platanoides, Populus tremula, Betula pendula and Picea abies. Under sterile conditions, at least three mixed samples were taken from the overlying trunks of each tree species and delivered frozen to the laboratory at each of the five stages of wood decomposition.
In parallel, DNA barcoding of fruiting body samples of xylobiont aphyllophoroid fungi collected from wood sample collection sites for metagenomic analysis was performed. Sixty new nucleotide sequences of the ITS1-5.8S-ITS2 region of the nrDNA were obtained, of which 20 sequences were deposited in the NCBI GenBank international database. Sixty new nucleotide sequences of the ITS1-5.8S-ITS2 region of nrDNA were obtained, of which 20 sequences were deposited in the NCBI GenBank. The complete nucleotide sequences of ITS1-5.8S-ITS2 nrDNA obtained and deposited in NCBI GenBank for the first time are mainly represented by taxa that were not previously reported in reference databases, which complicated their identification during metagenomic studies of woody substrates. Nucleotide sequences were obtained from authentic samples for little-known and rarely found in nature fungal species. A local database of reference DNA sequences of macromycetes was created, where for each nucleotide sequence information on the method of DNA isolation, primers used, as well as detailed information on the voucher herbarium specimen is given. In GBIF there was published information on 146 findings of aphyllophoroid fungi in the investigated sites of mass windthrow.
Physical and chemical characteristics of wood samples collected from the same stems of 8 tree species were analyzed (Khanina et al., 2023a,b). Characteristics of 247 samples at 5 stages of degradation were posted in GBIF. The analyses revealed that there was a significant increase in the concentrations of N, Mn, P, Cu, Zn and Ca during wood degradation. Significant differences in content and mass concentration between dead wood of different species were found for C, Mn, Zn, Mg, Ca and K. The following species were the leaders in the content of these elements: C - Picea and Tilia, Mn – Acer, Betula, Picea, Tilia; Zn - Betula and Populus; Mg – Acer, Ulmus; Ca – Ulmus; K – Tilia, Ulmus. It was revealed that for 14 years after mass windthrow there was a 1.5-fold decrease in deadwood stocks, while the stocks of elements in deadwood decreased 1.7-2.5 times for N, Mg and K and 2.7-3.5 times for C, P and Ca (Khanina et al., 2023c). A higher decomposition rate diffuse-sporous Angiosperms (birch, aspen, maple, linden) was found compared to ring-sporous Angiosperms (oak, ash, elm). The coniferous species spruce occupies an intermediate position between the decomposition rates of the deciduous species of the above groups.
In order to perform metagenomic analysis, the protocol for isolation of total DNA and amplification of variegated ITS regions of fungi was optimized. Metagenomic DNA (Qiagen DNeasy PowerSoil Pro Kit) was isolated from 144 wood samples of 8 species at 5 stages of decomposition. Variable regions of 16S rRNA from bacteria (V4) and ITS fungi (ITS1-2) were amplified. The obtained libraries were sequenced at the Skolkovo Institute of Science and Technology. Taxonomic classification of amplicon sequence variants (ASV) was performed based on the SILVA (Kim et al., 2019) and UNITE (Nilsson, 2019) databases using reference sequences of macromycete fruiting bodies collected from the investigated windthrow sites.
A total of 4717 different ITS1 sequence variants of fungi and 92262 sequence variants of the V4 regions of the 16S rRNA gene of bacteria were obtained from the samples. After downsampling, about 2500 and 22500 unique sequences remained in the fungal and bacterial communities, respectively. In all tree species there was an increase in alpha diversity of the fungal and bacterial communities between the initial and final stages of decomposition, at all stages of decomposition the bacterial community was more diverse than the fungal community. In the initial stages, the fungal community consisted on average of about 25 species per species (except spruce) with pronounced dominant representatives. The bacterial community of each tree species consisted typically of more than 200 species (except maple and birch). By the fourth and fifth stages of decomposition, all tree species had an average of more than 100 fungal species and from about 400 to more than 600 bacterial species. Some tree species showed a decline in the diversity of the investigated destructors at the second or third stage of decomposition. The concentration of amplicons from bacterial communities was more often higher at the last stage of decomposition than at the first one, while the fungal community often showed a peak at earlier stages (1-3) and a decline at the last one. According to our data, the maximum concentration of fungi and bacteria was characteristic of Tilia, and the minimum - of Quercus. In oak wood, bacteria appeared in noticeable numbers only by the third stage of decomposition, which probably correlates with its resistance to decomposition and, consequently, its active use in economy.
The fungal community was characterized by very high beta diversity, with the distance between samples of different woody species of the same decomposition stage being slightly smaller than the distance between samples of the same species but different stages, resulting in a more pronounced clustering of samples by decomposition stage than by tree species. During wood decomposition, the fungal community for some wood species changed dramatically, showing a shift in dominant representatives at the phylum level. Most fungal genera were observed only on samples of one species of one stage.
The bacterial community also showed very high beta diversity when assessed by Bray-Curtis distance. The similarity of the bacterial communities (like fungi, but for bacteria to a greater extent) clustered the samples more by stage than by tree species.
According to the survey of fruiting bodies of aphyllophoroid fungi, 127 species of basidial fungi were identified on dead wood of 8 tree species. The maximum species richness was observed on dead trunks of Picea (46 species). White rot fungi were dominant (74%), brown rot fungi accounted for 19%. Stems of different tree species did not differ significantly in the number of fungal species found on them. The composition of basidiomycetes on deadwood trunks of different tree species varied quite well, as shown by NMDS and PERMANOVA results. The species affiliation of tree trunks determined 45% of the variation in basidiomycete species composition. Additive partitioning of biodiversity showed that the contributions of all diversity components were significant, but the maximum contribution (81%) to the total gamma diversity of basidiomycetes was made by compositional diversity among tree species, which was statistically significantly higher than that expected under the null model.
In general, we obtained consistent estimates of the diversity of fungal communities identified from fruiting bodies and metagenomic analyses. The two analyses showed an increase in community diversity with increasing decomposition stage and the importance of the contribution of woody substrate diversity to the total gamma diversity of the fungal community of the analyzed site. The metagenomic analysis allowed a detailed assessment of the quantitative and qualitative change in the species diversity of destructor communities (bacteria and fungi) during wood decomposition of different tree species, suggesting specific features of diversity change during decomposition for each of the eight tree species examined.
The results of the study were presented at 9 scientific conferences. 14 articles were published, among them 3 in journals registered in WoS (Q1, Q3) and Scopus database; 8 in conference proceedings and 3 datasets.
References
- Khanina, L., Bobrovsky, M., Smirnov, V., Romanov, M. Wood decomposition, carbon, nitrogen, and pH values in logs of 8 tree species 14 and 15 years after a catastrophic windthrow in a mesic broad-leaved forest in the East European plain // Forest Ecology and Management. 2023a. Vol. 545. ID 121275. https://doi.org/10.1016/j.foreco.2023.121275
- Khanina, L.G., Smirnov, V.E., Bobrovskii, M.V. The elemental composition of dead wood in different tree species and stages of decay in the broad-leaved forests of the Kaluzhskie Zaseki State Nature Reserve // Russian Journal of Ecology. 2023b. Vol. 54. No. 7. P. 658–670. https://doi.org/10.1134/S106741362307007X
- Khanina, LG, Bobrovsky, MV, Smirnov, VE. Dynamics of Element Stocks in Deadwood and Soil After a Mass Windthrow in a Broad-Leaved Forest on Sandy Soils // Vestnik Tomskogo gosudarstvennogo universiteta. Biologiya = Tomsk State University Journal of Biology. 2023c. Vol. 62. P. 29-52. doi: 10.17223/19988591/62/2 (in Russian with English resume)
